Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum.
Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.
Lu
W P
WP
Department of Chemistry, University of Wisconsin, Milwaukee 53201.
Harder
S R
SR
Ragsdale
S W
SW
D000085:Acetates / Q000032:analysis / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000096:biosynthesis* / Q000302:isolation & purification; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction
D000085:Acetates; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D009097:Multienzyme Complexes; D000105:Acetyl Coenzyme A; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D008780:Methyltransferases